Haptens of aripiprazole

ABSTRACT

The invention relates to compounds of Formula I, wherein R 1 , R 2 , and R 3  are defined in the specification, useful for the synthesis of novel conjugates and immunogens derived from aripiprazole. The invention also relates to conjugates of an aripiprazole hapten and a protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/970,650, filed Aug. 20, 2013, and published Jun. 12, 2014, as US2014/0163206, which claims the benefit of U.S. Provisional ApplicationNo. 61/691,450, filed Aug. 21, 2012, all of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to the field of immunoassays for determining thepresence of aripiprazole in human biological fluids.

BACKGROUND OF THE INVENTION

Schizophrenia is a chronic and debilitating psychiatric disorderaffecting approximately 0.45-1% of the world's population (van Os, J.;Kapur, S. “Schizophrenia” Lancet 2009, 374, 635-645). The principalgoals of treatment are to achieve sustained remission from psychoticsymptoms, reduce the risk and consequences of relapse, and improvepatient functioning and overall quality of life. While many patientswith schizophrenia are able to achieve symptom stability with theavailable antipsychotic medications, poor adherence to medication is acommon reason for relapse with daily administered oral medications.Several studies (Abdel-Baki, A.; Ouellet-Plamondon, C.; Malla, A.“Pharmacotherapy Challenges in Patients with First-Episode Psychosis”Journal of Affective Disorders 2012, 138, S3-S14) investigating theoutcomes of non-compliance have shown that patients with schizophreniawho do not take their medication as prescribed have higher rates ofrelapse, hospital admission and suicide as well as increased mortality.It is estimated that 40 to 75% of patients with schizophrenia havedifficulty adhering to a daily oral treatment regimen (Lieberman, J. A.;Stroup, T. S.; McEvoy, J. P.; Swartz, M. S.; Rosenheck, R. A.; Perkins,D. O.; Keefe, R. S. E.; Davis, S. M.; Davis, C. E.; Lebowitz, B. D.;Severe, J.; Hsiao, J. K. “Effectiveness of Antipyschotic Drugs inPatients with Chronic Schizophrenia” New England Journal of Medicine2005, 353(12), 1209-1223). Therapeutic drug monitoring (TDM) is thequantification of serum or plasma concentrations of drugs, includinganti-psychotic drugs, for treatment monitoring and optimization. Suchmonitoring permits, for example, the identification of patients that arenot adhering to their medication regimen, that are not achievingtherapeutic doses, that are non-responsive at therapeutic doses, thathave suboptimal tolerability, that have pharmacokinetic drug-druginteractions, or that have abnormal metabolism resulting ininappropriate plasma concentrations. Considerable individual variabilityexists in the patient's ability to absorb, distribute, metabolize, andexcrete anti-psychotic drugs. Such differences can be caused byconcurrent disease, age, concomitant medication or geneticpeculiarities. Different drug formulations can also influence themetabolism of anti-psychotic drugs. TDM permits dose optimization forindividual patients, improving therapeutic and functional outcomes. TDMfurther permits a prescribing clinician to ensure compliance withprescribed dosages and achievement of effective serum concentrations.

To date, methods for determining the levels of serum or plasmaconcentrations of anti-psychotic drugs involve the use of liquidchromatography (LC) with UV or mass spectrometry detection, andradioimmunoassays (see, for example, Woestenborghs et al., 1990 “On theselectivity of some recently developed RIA's” in Methodological Surveysin Biochemistry and Analysis 20:241-246. Analysis of Drugs andMetabolites, Including Anti-infective Agents; Heykants et al., 1994 “ThePharmacokinetics of Risperidone in Humans: A Summary”, J Clin Psychiatry55/5, suppl:13-17; Huang et al., 1993 “Pharmacokinetics of the novelanti-psychotic agent risperidone and the prolactin response in healthysubjects”, Clin Pharmacol Ther 54:257-268). Radioimmunoas says detectone or both of risperidone and paliperidone. Salamone et al. in U.S.Pat. No. 8,088,594 disclose a competitive immunoassay for risperidoneusing antibodies that detect both risperidone and paliperidone but notpharmacologically inactive metabolites. The antibodies used in thecompetitive immunoassay are developed against a particular immunogen. IDLabs Inc. (London, Ontario, Canada) markets an ELISA for olanzapine,another anti-psychotic drug, which also utilizes a competitive format.The Instructions For Use indicate that the assay is designed forscreening purposes and intended for forensic or research use, and isspecifically not intended for therapeutic use. The Instructionsrecommend that all positive samples should be confirmed with gaschromatography/mass spectrometry (GC-MS), and indicate that the antibodyused detects olanzapine and clozapine (see ID Labs Inc., “InstructionsFor Use Data Sheet IDEL-F083”, Rev. Date Aug. 8, 2011). Some of thesemethods, namely HPLC and GC/MS, can be expensive and labor-intensive,and are generally only performed in large or specialty labs having theappropriate equipment.

A need exists for other methods for determining the levels ofanti-psychotic drugs, particularly methods that can be performed in aprescribing clinician's office (where the treatment for an individualpatient can be adjusted accordingly in a much more timely manner) and inother medical settings lacking LC or GC/MS equipment or requiring rapidtest results.

Aripiprazole is:

SUMMARY OF THE INVENTION

The subject invention provides compounds and conjugates that permit suchan improved method for determining the levels of the anti-psychotic drugaripiprazole.

The invention comprises compounds of Formula I:

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R³ is H, or W—(Y)_(p)-G; provided that two of R¹, R², R³ must be H, andfurther provided that R¹, R² and R³ may not all be H simultaneously;

wherein:

Z is selected from the group consisting of:

—N(R⁴)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-,

wherein:

W is selected from the group consisting of:

—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-;

R⁴ is H, an alkyl group, cycloalkyl group, aralkyl group or substitutedor unsubstituted aryl group;

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 0 or 1;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5.

The invention comprises conjugates of compounds of the invention withimmunogenic carriers such as proteins, and products produced by theprocess of contacting the compounds of the invention with immunogeniccarriers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Competition ELISA results generated with hybridoma 3C1;

FIG. 2 shows Competition ELISA results generated with hybridoma 3D7;

FIG. 3 shows the competitive immunoassay format used on a lateral flowassay device; and

FIGS. 4 and 5 show the results generated with capture antibodyaripiprazole clone 5C7 on the lateral flow assay device.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides compounds and conjugates that permit thedetermination of levels of anti-psychotic drugs. Such methods willpermit clinicians to evaluate objectively at an appointment how likelyit is that the worsening of a patient's symptoms may be due to lack ofadherence. Alternatively, if compliant, a clinician can consider adifferent treatment choice. Therapeutic drug monitoring, which isenabled by such methods, is key in identifying the most effectivetreatment options. Moreover, clinicians believe that such TDM will helpthem to move into a very different relationship with their patients,i.e., to move from a hypothetical discussion on treatment non-adherencetowards a more collaborative one by engaging patients to actively takeownership in optimizing their treatment regimen.

The development of the method requires first the synthesis of severalimmunogens, comprising a synthetic hapten linked to a protein. A haptenis a small molecule that can elicit an immune response when attached toa large carrier such as a protein. They are protein-free substances, ofmostly low molecular weight, which are not capable of stimulatingantibody formation alone, but which do react with antibodies. Ahapten-protein conjugate is able to stimulate the production ofantibodies. Specific antibody generation against small molecules isuseful for immunoassay development (Pharm Res. 1992, 9(11):1375-9,Annali Dell'Istituto Superiore di Sanita. 1991, 27(1):167-74, AnnaDell'Istituto Superiore di Sanita. 1991, 27(1):149-54, ImmunologyLetters. 1991, 28(1):79-83).

The invention comprises compounds of Formula I:

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R³ is H, or W—(Y)_(p)-G; provided that two of R¹, R², R³ must be H, andfurther provided that R¹, R² and R³ may not all be H simultaneously;

wherein:

Z is selected from the group consisting of:

—N(R⁴), —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-,

wherein:

W is selected from the group consisting of:

—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-;

R⁴ is H, an alkyl group, cycloalkyl group, aralkyl group or substitutedor unsubstituted aryl group;

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 0, or 1;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5.

Another embodiment of the invention comprises compounds of Formula I:

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R³ is H, provided that either R¹ or R² must be H, and further providedthat both R¹ and R² may not be H simultaneously;

wherein:

Z is selected from the group consisting of:

—N(R⁴)—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-,

R⁴ is H, an alkyl group, cycloalkyl group, aralkyl group or substitutedor unsubstituted aryl group;

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 0, or 1;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5.

Another embodiment of the invention comprises compounds of Formula I:

wherein:

R1 is H, or CH₂NH—(Y)_(p)-G;

R2 is H, or NH—(Y)_(p)-G;

R³ is H, provided that either R¹ or R² must be H, and further providedthat both R¹ and R² may not be H simultaneously;

wherein:

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 1.

Another embodiment of the invention comprises compounds of Formula I:

wherein:

R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H,

NH₂, or NHC(O)(CH₂)_(n)CO₂H; provided that either

R¹ or R² must be H, and further provided that both R¹ and R² may not beH simultaneously;

R³ is H;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5.

In another embodiment of the invention:

R¹ is H, CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H, NH₂, orNHC(O)(CH₂)_(n)CO₂H; provided that either R¹ or R² must be H, andfurther provided that both R¹ and R² may not be H simultaneously;

R³ is H;

m is 1, 2 or 3;

n is 1, 2 or 3.

In another embodiment of the invention:

R¹ is H, CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H, NH₂, or NHC(O)(CH₂)_(n)CO₂H; provided that either R¹ or R² mustbe H, and further provided that both R¹ and R² may not be Hsimultaneously;

R³ is H;

m is 2;

n is 2.

Another embodiment of the invention is a compound selected from thegroup consisting of:

A preferred embodiment of the invention is the compound:

Another preferred embodiment of the invention is the compound:

The invention further provides conjugates of the above compounds with animmunogenic carrier.

Another embodiment of the invention is thus a conjugate of the compoundof Formula 1

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R³ is H, or W—(Y)_(p)-G; provided that two of R¹, R², R³ must be H, andfurther provided that R¹, R² and R³ may not all be H simultaneously;

wherein:

Z is selected from the group consisting of:

—NR⁴—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, alkylcarbonyl-,

—R⁴ is H, an alkyl group, cycloalkyl group or substituted orunsubstituted aryl group;

wherein:

W is selected from the group consisting of:

—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-;

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 0, or 1;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5; and an immunogenic carrier.

Another embodiment of the invention is a conjugate of the compound ofFormula 1 wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

provided that either R¹ or R² must be H, and further provided that bothR¹ and R² may not be H simultaneously;

R³ is H;

wherein:

Z is selected from the group consisting of:

—NR⁴—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-,

R⁴ is H, an alkyl group, cycloalkyl group or substituted orunsubstituted aryl group;

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 0 or 1;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5; and an immunogenic carrier.

Another embodiment of the invention is a conjugate of the compound ofFormula 1

wherein:

R¹ is H, or CH₂NH—(Y)_(p)-G;

R² is H, or NH—(Y)_(p)-G;

R³ is H, provided that either R¹ or R² must be H, and further providedthat both R¹ and R² may not be H simultaneously;

wherein:

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 1; and an immunogenic carrier.

Another embodiment of the invention is a conjugate of the compound ofFormula 1

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H;

provided that either R¹ or R² must be H, and further provided that bothR¹ and R² may not be H simultaneously;

R³ is H;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5; and an immunogenic carrier.

Another embodiment of the invention is a conjugate of the compound ofFormula 1

wherein:

R¹ is H, CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H, NH₂, or NHC(O)(CH₂)_(n)CO₂H; provided that either R¹ or R² mustbe H, and further provided that both R¹ and R² may not be Hsimultaneously;

m is 1, 2 or 3;

n is 1, 2 or 3; and an immunogenic carrier.

Another embodiment of the invention is a conjugate of the compound ofFormula 1

wherein:

R¹ is H, CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H, NH₂, or NHC(O)(CH₂)_(n)CO₂H; provided that either R¹ or R² mustbe H, and further provided that both R¹ and R² may not be Hsimultaneously;

m is 2;

n is 2; and an immunogenic carrier.

Another embodiment of the invention is a conjugate of a compoundselected from the group consisting of:

and an immunogenic carrier.

A preferred embodiment of the invention is any of the above conjugateswherein the immunogenic carrier is a protein.

A preferred embodiment of the invention is any of the above conjugates,wherein said protein is keyhole limpet hemocyanin, ovalbumin or bovinethyroglobulin.

The invention also provides products formed from the process ofcontacting the above compounds with an immunogenic carrier.

Another embodiment of the invention is thus a product formed from theprocess of contacting a compound of Formula I

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R³ is H, or W—(Y)_(p)-G; provided that two of R¹, R², R³ must be H, andfurther provided that R¹, R² and R³ may not all be H simultaneously;

wherein:

Z is selected from the group consisting of:

—NR⁴—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, alkylcarbonyl-,

—R⁴ is H, an alkyl group, cycloalkyl group or substituted orunsubstituted aryl group;

wherein:

W is selected from the group consisting of:

—C(O)—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-;

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 0, or 1;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5; with an immunogenic carrier.

Another embodiment of the invention is a product formed from the processof contacting a compound of Formula I

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H, or Z—(Y)_(p)-G;

provided that either R¹ or R² must be H, and further provided that bothR¹ and R² may not be H simultaneously;

R³ is H;

wherein:

Z is selected from the group consisting of:

—NR⁴—, —O—, —S—, -alkyl-, -alkoxyalkyl-, -aminoalkyl-, -thioalkyl-,-heteroalkyl-, -alkylcarbonyl-,

R⁴ is H, an alkyl group, cycloalkyl group or substituted orunsubstituted aryl group;

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 0, or 1;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5; with an immunogenic carrier.

Another embodiment of the invention is a product formed from the processof contacting a compound of Formula I

wherein:

R¹ is H, or CH₂NH—(Y)_(p)-G;

R² is H, or NH—(Y)_(p)-G;

R³ is H, provided that either R¹ or R² must be H, and further providedthat both R¹ and R² may not be H simultaneously;

wherein:

Y is an organic spacer group;

G is a functional linking group capable of binding to a carrier;

p is 1; with an immunogenic carrier.

Another embodiment of the invention is a product formed from the processof contacting a compound of Formula I

wherein:

R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H, NH₂, NHC(O)(CH₂)_(m)CO₂H;

provided that either R¹ or R² must be H, and further provided that bothR¹ and R² may not be H simultaneously;

R³ is H;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5; with an immunogenic carrier.

Another embodiment of the invention is a product formed from the processof contacting a compound of Formula I

wherein:

R¹ is H, CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H, NH₂, or NHC(O)(CH₂)_(n)CO₂H; provided that either R¹ or R² mustbe H, and further provided that both R¹ and R² may not be Hsimultaneously;

m is 1, 2 or 3;

n is 1, 2 or 3; with an immunogenic carrier.

Another embodiment of the invention is a product formed from the processof contacting a compound of Formula I

wherein:

R¹ is H, CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H;

R² is H, NH₂, or NHC(O)(CH₂)_(n)CO₂H; provided that either R¹ or R² mustbe H, and further provided that both R¹ and R² may not be Hsimultaneously;

m is 2;

n is 2; with an immunogenic carrier.

A preferred embodiment of the invention is a product formed from theprocess of contacting the compound

with an immunogenic carrier.

A preferred embodiment of the invention is a product formed from theprocess of contacting the compound

with an immunogenic carrier.

A preferred embodiment of the invention is a product formed from theprocess of contacting the compound

wherein m is 2 or 3; with an immunogenic carrier.

A preferred embodiment of the invention is any of the above productswherein the immunogenic carrier is a protein.

A preferred embodiment of the invention is any of the above products,wherein said protein is keyhole limpet hemocyanin, ovalbumin or bovinethyroglobulin.

ABBREVIATIONS

Herein and throughout the application, the following abbreviations maybe used.

-   AIBN azobisisobutyronitrile-   AMAS N-(α-maleimidoacetoxy)succinimide ester-   BTG bovine thyroglobulin-   Bu₃N tributylamine-   DMF N,N-dimethylformamide-   EDTA ethylenediaminetetraaceticacid-   EtOH ethyl alcohol-   KLH keyhole limpet hemocyanin-   NBS N-bromo succinimide-   SATA N-succinimidyl S-acetylthioacetate-   THF tetrahydrofuran-   TFA trifluoroacetic acid-   DCC dicyclohexylcarbodiimide-   DIC diisopropylcarbodiimide-   DMAP N,N-dimethyl-4-aminopyridine-   EDC 1-ethyl-3(3-dimethylaminopropyl) carbodiimidehydrochloride-   NHS N-hydroxysuccinimide-   TFP Tetrafluorophenyl-   PNP p-nitrophenyl-   TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    tetrafluoroborate-   HOBT N-Hydroxybenzotriazole-   DEPBT 3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one-   BOP-Cl DTT Bis(2-oxo-3-oxazolidinyl)phosphonic chloride    dithioerythritol

DEFINITIONS

The term “conjugate” refers to any substance formed from the joiningtogether of separate parts. Representative conjugates in accordance withthe present invention include those formed by the joining together of asmall molecule, such as the compounds of Formula I, and a largemolecule, such as a carrier or a polyamine polymer, particularly aprotein. In the conjugate the small molecule may be joined at one ormore active sites on the large molecule.

The term “hapten” refers to a partial or incomplete antigen. A hapten isa protein-free substance, which is not capable of stimulating antibodyformation, but which does react with antibodies. The antibodies areformed by coupling a hapten to a high molecular weight immunogeniccarrier, and then injecting this coupled product, i.e., an immunogen,into a human or animal subject.

The term “immunogen” refers to a substance capable of eliciting,producing, or generating an immune response in an organism.

An “immunogenic carrier,” as used herein, is an immunogenic substance,commonly a protein, that can join at one or more positions with haptens,thereby enabling the production of antibodies that can bind specificallywith these haptens. Examples of immunogenic carrier substances include,but are not limited to, proteins, glycoproteins, complexpolyamino-polysaccharides, particles, and nucleic acids that arerecognized as foreign and thereby elicit an immunologic response fromthe host. The polyamino-polysaccharides may be prepared frompolysaccharides using any of the conventional means known for thispreparation.

Various protein types may be employed as immunogenic carriers, includingwithout limitation, albumins, serum proteins, lipoproteins, etc.Illustrative proteins include bovine serum albumin, keyhole limpethemocyanin, egg ovalbumin, bovine thyroglobulin, fraction V human serumalbumin, rabbit albumin, pumpkin seed globulin, diphtheria toxoid,tetanus toxoid, botilinus toxin, succinylated proteins, and syntheticpoly(aminoacids) such as polylysine.

Immunogenic carriers can also include poly amino-polysaccharides, whichare a high molecular weight polymers built up by repeated condensationsof monosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums such as gum arabic, agar, and so forth. Thepolysaccharide also contains poly(amino acid) residues and/or lipidresidues.

The immunogenic carrier can also be a poly(nucleic acid) either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The immunogenic carrier can also include solid particles. The particlesare generally at least about 0.02 microns (μm) and not more than about100 μm, and usually about 0.05 μm to 10 μm in diameter. The particle canbe organic or inorganic, swellable or non-swellable, porous ornon-porous, optimally of a density approximating water, generally fromabout 0.7 to 1.5 g/mL, and composed of material that can be transparent,partially transparent, or opaque. The particles can be biologicalmaterials such as cells and microorganisms, including non-limitingexamples such as erythrocytes, leukocytes, lymphocytes, hybridomas,Streptococcus, Staphylococcus aureus, E. coli, and viruses. Theparticles can also be comprised of organic and inorganic polymers,liposomes, latex, phospholipid vesicles, or lipoproteins.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “analogue” of a chemical compound refers to a chemical compoundthat contains a chain of carbon atoms and the same particular functionalgroups as a reference compound, but the carbon chain of the analogue islonger or shorter than that of the reference compound.

A “label,” “detector molecule,” “reporter” or “detectable marker” is anymolecule which produces, or can be induced to produce, a detectablesignal. The label can be conjugated to an analyte, immunogen, antibody,or to another molecule such as a receptor or a molecule that can bind toa receptor such as a ligand, particularly a hapten or antibody.Non-limiting examples of labels include radioactive isotopes (e.g.,¹²⁵I), enzymes (e.g., β-galactosidase, peroxidase), enzyme fragments,enzyme substrates, enzyme inhibitors, coenzymes, catalysts, fluorophores(e.g., rhodamine, fluorescein isothiocyanate or FITC, or Dylight 649),dyes, chemiluminescers and luminescers (e.g., dioxetanes, luciferin), orsensitizers.

As used herein, a “spacer” refers to a portion of a chemical structurewhich connects two or more substructures such as haptens, carriers,immunogens, labels or binding partners through a functional linkinggroup. These spacer groups are composed of the atoms typically presentand assembled in ways typically found in organic compounds and so may bereferred to as “organic spacing groups”. The chemical building blocksused to assemble the spacers will be described hereinafter in thisapplication. Among the preferred spacers are straight or branched,saturated or unsaturated carbon chains. These carbon chains may alsoinclude one or more heteroatoms within the chain, one or moreheteroatoms replacing one or more hydrogens of any carbon atom in thechain, or at the termini of the chains. By “heteroatoms” is meant atomsother than carbon which are chosen from the group consisting of oxygen,nitrogen, phosphorous and sulfur, wherein the nitrogen, phosphorous andsulfur atoms may exist in any oxidation state and may have carbon orother heteroatoms bonded to them. The spacer may also include cyclic oraromatic groups as part of the chain or as a substitution on one of theatoms in the chain.

The number of atoms in the spacing group is determined by counting theatoms other than hydrogen. The number of atoms in a chain within aspacing group is determined by counting the number of atoms other thanhydrogen along the shortest route between the substructures beingconnected. Preferred chain lengths are between 1 to 20 atoms.

A “functional linking group” refers to a reactive group that is presenton a hapten and may be used to provide an available reactive sitethrough which the hapten portion may be coupled to another moietythrough formation of a covalent chemical bond to produce a conjugate ofa hapten with another moiety (such as a label or carrier). The haptenmay be linked in this way to a moiety such as biotin to form acompetitive binding partner for the hapten.

Spacer groups may be used to link the hapten to the carrier. Spacers ofdifferent lengths allow one to attach the hapten with differingdistances from the carrier for presentation to the immune system of theanimal or human being immunized for optimization of the antibodyformation process. Attachment to different positions in the haptenmolecule allows the opportunity to present specific sites on the haptento the immune system to influence antibody recognition. The spacer maycontain hydrophilic solubilizing groups to make the hapten derivativemore soluble in aqueous media. Examples of hydrophilic solubilizinggroups include but are not limited to polyoxyalkyloxy groups, forexample, polyethylene glycol chains; hydroxyl, carboxylate and sulfonategroups.

The term “nucleophilic group” or “nucleophile” refers to a species thatdonates an electron-pair to form a chemical bond in a reaction. The term“electrophilic group” or “electrophile” refers to a species that acceptsan electron-pair from a nucleophile to form a chemical bond in areaction.

The term “substituted” refers to substitution of an atom or group ofatoms in place of a hydrogen atom on a carbon atom in any position onthe parent molecule. Non limiting examples of substituents includehalogen atoms, amino, hydroxy, carboxy, alkyl, aryl, heteroalkyl,heteroaryl, cyano, alkoxy, nitro, aldehyde and ketone groups.

The term “alkyl” refers to saturated or unsaturated linear and branchedchain radicals of up to 12 carbon atoms, unless otherwise indicated, andis specifically intended to include radicals having any degree or levelof saturation. Alkyl includes, but is not limited to, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, hexyl, isohexyl, heptyl, octyl, 2,2,4-trimethylpentyl, nonyl,decyl, undecyl and dodecyl.

The term “cycloalkyl” refers to a saturated or partially unsaturatedmonocyclic or bicyclic hydrocarbon ring radical composed of from 3 to 10carbon atoms. Alkyl substituents may optionally be present on the ring.Examples include cyclopropyl, 1,1-dimethyl cyclobutyl,1,2,3-trimethylcyclopentyl, cyclohexyl and cyclohexenyl.

The term “heteroatom” refers to a nitrogen atom, an oxygen atom, aphosphorous atom or a sulfur atom wherein the nitrogen, phosphorous andsulfur atoms can exist in any allowed oxidation states.

The term “heteroalkyl” refers to an alkyl group that includes one ormore heteroatoms within the chain, one or more heteroatoms replacing oneor more hydrogens of any carbon atom in the chain, or at termini of thechains.

The term “heterocyclyl” refers to a nonaromatic (i.e. saturated orpartially unsaturated) ring composed of from 3 to 7 carbon atoms and atleast one heteroatom selected from N, O or S. Alkyl substituents mayoptionally be present on the ring. Examples include tetrahydrofuryl,dihydropyranyl, piperidyl, 2,5-dimethypiperidyl, morpholinyl,piperazinyl, thiomorpholinyl, pyrrolidinyl, pyrrolinyl, pyrazolidinyl,pyrazolinyl, imidazolidinyl and imidazolinyl.

The term “hydroxyalkyl” refers to at least one hydroxyl group bonded toany carbon atom along an alkyl chain.

The term “aminoalkyl” refers to at least one primary or secondary aminogroup bonded to any carbon atom along an alkyl chain.

The term “alkoxy” refers to straight or branched chain radicals of up to12 carbon atoms, unless otherwise indicated, bonded to an oxygen atom.Examples include but are not limited to methoxy, ethoxy, propoxy,isopropoxy and butoxy.

The term “alkoxyalkyl” refers to at least one alkoxy group bonded to anycarbon atom along an alkyl chain.

The term “polyalkoxyalkyl” refers to long-chain alkoxy compounds andincludes polyethylene glycols of discreet or monodispersed sizes.

The term “thioalkyl” refers to at least one sulfur group bonded to anycarbon atom along an alkyl chain. The sulfur group may be at anyoxidation state and includes sulfoxides, sulfones and sulfates.

The term “carboxyalkyl” refers to at least one carboxylate group bondedto any carbon atom along an alkyl chain. The term “carboxylate group”includes carboxylic acids and alkyl, cycloalkyl, aryl or aralkylcarboxylate esters.

The term “alkylcarbonyl” refers to a group that has a carbonyl groupbonded to any carbon atom along an alkyl chain.

The term “heteroaryl” refers to 5- to 7-membered mono- or 8- to10-membered bicyclic aromatic ring radicals, any ring of which mayconsist of from one to four heteroatoms selected from N, O or S wherethe nitrogen and sulfur atoms can exist in any allowed oxidation state.Examples include benzimidazolyl, benzothiazolyl, benzothienyl,benzoxazolyl, furyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl,pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl,thiazolyl and thienyl.

The term “aryl” refers to monocyclic or bicyclic aromatic ring radicalscontaining from 6 to 12 carbons in the ring. Alkyl substituents mayoptionally be present on the ring. Examples include phenyl, biphenyl andnapththalene.

The term “aralkyl” refers to a C₁₋₆ alkyl group containing an arylsubstituent. Examples include benzyl, phenylethyl or 2-naphthylmethyl.

The term “acyl” refers to the group —C(O)R_(a), where R_(a) is hydrogen,alkyl, cycloalkyl, heteroalkyl, aryl, aralkyl and heteroaryl. An“acylating agent” adds the —C(O)R_(a) group to a molecule.

The term “sulfonyl” refers to the group —S(O)₂R_(b), where R_(b) ishydrogen, alkyl, cycloalkyl, heteroalkyl, haloalkyl, aryl, aralkyl andheteroaryl. A “sulfonylating agent” adds the —S(O)₂R_(a) group to amolecule.

Spacers bearing reactive functional linking groups for the attachment ofhaptens to carrier moieties may be prepared by a wide variety ofmethods. The spacer may be formed using a molecule that isdifferentially functionalized or activated with groups at either end toallow selective sequential reaction with the hapten and the carrier, butthe same reactive moiety may also be used at both ends. The groupsselected for reaction with the hapten and the functional linking groupto be bound to the carrier are determined by the type of functionalityon the hapten and the carrier that the hapten is to be bonded with.Spacers and methods of attachment to haptens and carriers include butare not limited to those described by Brinkley, M., A., BioconjugateChem. 1992, 3:2-13, Hermanson, Greg T., Bioconjugate Techniques,Academic Press, London, Amsterdam, Burlington, Mass., USA, 2008 andThermo Scientific Pierce Crosslinking Technical Handbook; available fordownload or hard copy request from Thermo Scientific 3747 N Meridian Rd,Rockford, Ill. USA 61101, ph 800-874-3723 or at:http://www.piercenet.com/ and references within. Many differentiallyactivated molecules for formation of spacer groups are commerciallyavailable from vendors, for example Thermo Scientific.

For haptens bearing an amino group, modes of attachment of the spacer tothe hapten include reaction of the amine on the hapten with a spacerbuilding block bearing an acyl halide or active ester. “Active esters”are defined as esters that undergo reaction with a nucleophilic group,for example an amino group, under mild conditions to form a stablelinkage. A stable linkage is defined as one that remains intact underconditions of further use, for example subsequent synthetic steps, useas an immunogen, or in a biochemical assay. A preferred example of astable linkage is an amide bond. Active esters and methods of formationare described by Benoiton, N. L., in Houben-Weyl, Methods of OrganicChemistry, Thieme Stuttgart, New York, vol E22 section 3.2:443 andBenoiton, N. L., Chemistry of Peptide Synthesis, Taylor and Francis, NY,2006. Preferred active esters include p-nitrophenyl ester (PNP),N-hydroxysuccinimide ester (NHS) and tetrafluorophenyl ester (TFP). Acylhalides may be prepared by many methods known to one skilled in the artfor example, reaction of the carboxylic acid with thionyl chloride oroxalyl chloride, see: Fieser, L. F. and Fieser, M. Reagents for OrganicSynthesis, John Wiley and Sons, NY, 1967 and references within. Thesemay be converted to other active esters such as p-nitrophenyl esters(PNP) which may also be used in active bi-functional spacers asdescribed by Wu et. al, Organic Letters, 2004, 6 (24):4407.N-hydroxysuccinimide (NHS) esters may be prepared by reaction ofN,N-disuccinimidyl carbonate (CAS 74124-79-1) with the carboxylic acidof a compound in the presence of an organic base such as triethylamineor diisopropylethylamine in an aprotic solvent under anhydrousconditions as described in example 35 of WO2012012595 or by usingN-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) or otherdehydrating agent, under anhydrous conditions. Tetrafluorophenyl esters(TFP) may be prepared by reaction of carboxylic acids with2,3,5,6-tetrafluorophenyltrifluoroacetate in the presence of an organicbase such as triethylamine or diisopropylethylamine in an aproticsolvent under anhydrous conditions as reported by Wilbur, et. al,Bioconjugate Chem., 2004, 15(1):203. One skilled in the art willrecognize that spacers shown in Table 1, among others, can be obtainedusing known methods and attached to amino-bearing haptens utilizingroutine optimization of reaction conditions. These spacers allowattachment of the hapten to a thiol group on a carrier.

TABLE 1

Reasonable values for m and n are between 1 and 10

Direct coupling of the amine on the hapten and a carboxylic acidfunctionality on the spacer building block in the presence of a couplingagent may also be used as a mode of attachment. Preferred reagents arethose typically used in peptide synthesis. Peptide coupling reagentsinclude but are not limited toO-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU, CAS #125700-67-6), see: Pruhs, S., Org. Process. Res. Dev. 2006,10:441; N-Hydroxybenzotriazole (HOBT, CAS #2592-95-2) with acarbodiimide dehydrating agent, for example N—N-dicyclohexylcarbodiimide(DCC), diisopropylcarbodiimide (DIC), or1-ethyl-3(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), see:Kinig W., Geiger, R. Chem. Ber., 1970, 103 (3):788;3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one (DEPBT,CAS#165534-43-0), see: Liu, H. et. al., Chinese Chemical Letters, 2002,13(7):601; Bis(2-oxo-3-oxazolidinyl)phosphonic chloride; (BOP-Cl,CAS#68641-49-6), see: Diago-Meseguer, J et. al. Synthesis, 1980,7:547-51 and others described in detail by Benoiton in Chemistry ofPeptide Synthesis, CRC Press, Boca Raton, Fla., 2005, Chapter 2, and thetechnical bulletin provided by Advanced Automated Peptide ProteinTechnologies (aapptec), 6309 Shepardsville Rd., Louisville Ky. 40228, ph888 692 9111; www.aapptec.com, and references within. These methodscreate a stable amide linkage attaching the hapten to the spacer.Examples of spacers that can be obtained using known methods andattached to amino-bearing haptens utilizing routine optimization ofreaction conditions employing the methods described and cited above areshown, but not limited to those in Table 2. These spacers allowattachment of the hapten to a thiol group on a carrier.

TABLE 2

Spacers may also be constructed in a step-wise fashion by sequentialattachment of appropriate chemical groups to the hapten including thestep of forming the functional linking group that is capable of bindingto the carrier. See illustrative examples under General ReactionSchemes.

Additionally, when the hapten has a nucleophilic group, for example athiol group, an amino group or a hydroxyl group which will become thepoint of attachment of the spacer, the spacer may also be constructed byalkylation of the thiol, amine or hydroxyl group. Any alkyl group thatis appropriately substituted with a moiety capable of undergoing asubstitution reaction, for example, an alkyl halide, or sulfonic acidester such as p-Toluenesulfonate, may be used to attach the spacer. Manyexamples of alkylation reactions are known to one skilled in the art andspecific examples may be found in the general chemical literature andoptimized through routine experimentation. A discussion of alkylationreactions with many references can be found in Chapter 10 of March'sAdvanced Organic Chemistry, Smith, M. B., and March, J., John Wiley &sons, Inc. NY, 2001. Other linkages may also be employed such asreaction of the nucleophilic moiety, for example an amine, on the haptenwith an isocyanate to form a urea or reaction with an isothiocyanate toform a thiourea linkage, see: Li, Z., et. al., Phosphorus, Sulfur andSilicon and the Related Elements, 2003, 178(2):293-297. Spacers may beattached to haptens bearing hydroxyl groups via reaction with isocyanategroups to form carbamate or urethane linkages. The spacer may bedifferentially activated with the isocyanate functional group on one endand a functional linking group capable of reacting with the carrier,see: Annunziato, M. E., Patel, U.S., Ranade, M. and Palumbo, P. S.,Bioconjugate Chem., 1993, 4:212-218.

For haptens bearing a carboxylic acid group, modes of attachment of aspacer portion to the hapten include activation of the carboxylic acidgroup as an acyl halide or active ester, examples of which are shown inTable 3, preparation of which are described previously, followed byreaction with an amino (—NH₂—), hydrazino (—NH—NH₂—), hydrazido(—C(O)—NH—NH₂—) or hydroxyl group (—OH) on the spacer portion to form anamide, hydrazide, diacylhydrazine or ester linkage, or direct couplingof the carboxylic acid group with an amino group on the spacer portionor directly on the carrier with a peptide coupling reagent and/orcarbodiimide dehydrating reagent, described previously, examples ofwhich are shown in Tables 4 and 5. Procedures found in references citedpreviously for formation of activated esters and use of peptide couplingagents may be employed for attachment of carboxylic acid-bearing haptensto spacer building blocks and protein carriers with available aminogroups utilizing routine optimization of reaction conditions.

TABLE 3

TABLE 4

TABLE 5

Other electrophilic groups may be present on the hapten to attach thespacer, for example, a sulfonyl halide

or electrophilic phosphorous group, for example:

See: Malachowski, William P., Coward, James K., Journal of OrganicChemistry, 1994, 59 (25):7616

or:

R_(c) is alkyl, cycloalkyl, aryl, substituted aryl, aralkyl.

See: Aliouane, L., et. al, Tetrahedron Letters, 2011, 52(28):8681.

Haptens that bear aldehyde or ketone groups may be attached to spacersusing methods including but not limited to reaction with a hydrazidegroup H₂N—NH—C(O)— on the spacer to form an acylhydrazone, see: Chamow,S. M., Kogan, T. P., Peers, D. H., Hastings, R. C., Byrn, R. A. andAskenaszi, A., J. Biol. Chem., 1992, 267(22): 15916. Examples ofbifunctional hydrazide spacer groups that allow attachment to a thiolgroup on the carrier are shown in Table 6.

TABLE 6

Haptens may also contain thiol groups which may be reacted with thecarrier provided that the carrier has been modified to provide a groupthat may react with the thiol. Carrier groups may be modified by methodsincluding but not limited to attachment of a group containing amaleimide functional group by reaction of an amino group on the carrierwith N-Succinimidyl maleimidoacetate, (AMAS, CAS #55750-61-3),Succinimidyl iodoacetate (CAS#151199-81-4), or any of the bifunctionalspacer groups shown in Table 1 to introduce a group which may undergo areaction resulting in attachment of the hapten to the carrier.

The functional linking group capable of forming a bond with the carriermay be any group capable of forming a stable linkage and may be reactiveto a number of different groups on the carrier. The functional linkinggroup may preferably react with an amino group, a carboxylic acid groupor a thiol group on the carrier, or derivative thereof. Non-limitingexamples of the functional linking group are a carboxylic acid group,acyl halide, active ester (as defined previously), isocyanate,isothiocyanate, alkyl halide, amino group, thiol group, maleimide group,acrylate group (H₂C═CH—C(O)—) or vinyl sulfone group H₂C═CH—SO₂—) See:Park, J. W., et. al., Bioconjugate Chem., 2012, 23(3): 350. Thefunctional linking group may be present as part of a differentiallyactivated spacer building block that may be reacted stepwise with thehapten and the resulting hapten derivative may then be reacted with thecarrier. Alternatively, the hapten may be derivatized with a spacer thatbears a precursor group that may be transformed into the functionallinking group by a subsequent reaction. When the functional linkinggroup on the spacer is an amine or a carboxylic acid group, the couplingreaction with the carboxylic acid group or amine on the carrier may becarried out directly through the use of peptide coupling reagentsaccording to procedures in the references cited above for thesereagents.

Particular disulfide groups, for example, pyridyldisulfides, may be usedas the functional linking group on the spacer which may undergo exchangewith a thiol group on the carrier to from a mixed disulfide linkage,see: Ghetie, V., et al., Bioconjugate Chem., 1990, 1:24-31. Thesespacers may be attached by reaction of the amine-bearing hapten with anactive ester which is attached to a spacer bearing the pyridyldisulfidegroup, examples of which include but are not limited to those shown inTable 7.

TABLE 7

Most often the carrier is a protein and the ε-amino groups of the lysineresidues may be used for attachment, either directly by reaction with anamine-reactive functional linking group or after derivitization with athiol-containing group, including N-Succinimidyl S-Acetylthioacetate,(SATA, CAS 76931-93-6), or an analogue thereof, followed by cleavage ofthe actetate group with hydroxylamine to expose the thiol group forreaction with the functional linking group on the hapten. Thiol groupsmay also be introduced into the carrier by reduction of disulfide bondswithin protein carriers with mild reducing reagents including but notlimited to 2-mercaptoethylamine, see: Bilah, M., et. al.,Bioelectrochemistry, 2010, 80(1):49, phosphine reagents, see: Kirley, T.L., Analytical Biochemistry, 1989, 180(2):231 or dithioerythritol (DTT,CAS 3483-12-3) Cleland, W., Biochemistry, 1964, 3:480-482.

General Reaction Schemes

Representative compounds of the present invention can be synthesized inaccordance with the general synthetic methods described below. Compoundsof Formula I can be prepared by methods known to those who are skilledin the art. The following reaction schemes are only meant to representexamples of the invention and are in no way meant to be a limit of theinvention.

The hapten of Example 1 may be elaborated with spacers by reaction witha cyclic anhydride compound, such as succinic anhydride or glutaricanhydride, as shown in Scheme 1. The reaction may be carried out in asolvent such as THF, at room temperature, overnight.

The hapten of Example 2 may be elaborated with spacers by reaction witha cyclic anhydride compound, such as succinic anhydride or glutaricanhydride, as shown in Scheme 2. The reaction may be carried out in asolvent such as pyridine, and heated to about 110° C. in a microwaveoven for 3-6 hours.

Haptens which terminate in an alkyl amine group, such as Example 1 maybe further functionalized with a maleimide group. Those skilled in theart will recognize that the same methodology will be applicable to otheralkyl amino derivatives of aripiprazole. Reaction of the aripiprazolederived amine with alkyl-maleimide functionalizing group, such as2,5-dioxopyrrolidin-1-yl2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate, in a solvent such asDMF, in the presence of a base, such as tributyl amine, at 20° C., forone hour generates haptens of aripiprazole with a maleimide spacer.

Spacers on haptens may be extended as shown in Scheme 4. Haptens withspacers bearing a carboxylic acid functionality may be dissolved in asuitable solvent, such as dichloromethane, under inert atmosphere, andtreated with N-t-butoxycarbonylpiperazine and an appropriate base, suchas diisopropylethylamine. The solution may then be treated with diethylcyanophosphonate to install a piperazine moiety onto the spacer.Deprotection of the piperazine may be accomplished with trifluoroaceticacid or other methods known in the art. Reaction with a cyclic anhydridegives compounds of Formula I where R¹ is

Spacers on haptens may also be extended as shown in Scheme 5. Haptenswith spacers bearing carboxylic acid functionality may be dissolved in asuitable solvent, such as dichloromethane, under inert atmosphere, andtreated with N-t-butoxycarbonylpiperazine and an appropriate base, suchas diisopropylethylamine. The solution may then be treated with diethylcyanophosphonate to install a piperazine moiety onto the spacer.Deprotection of the piperazine may be accomplished with trifluoroaceticacid or other methods known in the art. Reaction with a cyclic anhydridegives compounds of Formula I where R² is

Haptens may also be generated directly from the parent moleculearipiprazole by either acylation or alkylation of the quinolinonenitrogen. Scheme 6 depicts a synthetic route in which an acyl group maybe appended to aripiprazole by reaction with the acid chloride of4-chlorobutyric acid using N,N-dimethyl-4-aminopyridine (DMAP) as acatalyst in the presence of a base such as pyridine in an aproticsolvent, for example N,N-dimethylformamide, see: Example 5,US20110230520. Nucleophilic substitution of the chloride byN-methyl-β-alanine methyl ester may be carried out in the presence ofsodium iodide and a base, for example potassium carbonate in a dipolaraprotic solvent such as N,N-dimethylformamide, see: Penning, T., D., et.al., J. Med Chem, 2002, 45:3482. Hydrolysis of the ester group usingstandard methods known to one skilled in the art, such as exposure toaqueous base, yields a carboxy-functionalized hapten which may befurther elaborated using the methods described previously, one exampleof which is depicted in Scheme 9 below, to provide a suitablyfunctionalized compound for attachment to an immunogenic carrier.

Scheme 7 illustrates a mode of attachment of an alkyl group to thenitrogen of the quinolinone group of aripiprazole using standardalkylation chemistry. An iodo compound, for examplemethyl-4-iodobutyrate may be reacted with aripiprazole in the presenceof a base such as cesium carbonate in a dipolar aprotic solvent such asN,N-dimethylformamide using the method of Example 6 in US20120004165.Hydrolysis of the ester group using standard methods known to oneskilled in the art, such as exposure to aqueous base, yields acarboxy-functionalized hapten which may be further elaborated using themethods described previously, one example of which is depicted in Scheme9 below, to provide a suitably functionalized compound for attachment toan immunogenic carrier.

Maleimide functionalized haptens may be conjugated to proteins accordingto the method shown in Scheme 8. Activation of protein lysine residuesby acylation of the epsilon-nitrogen with N-succinimidylS-acetylthioacetate (SATA), followed by subsequent hydrolysis of theS-acetyl group with hydroxylamine produces a nucleophilic sulfhydrylgroup. Conjugation of the sulfhydryl activated protein with themaleimide derivatized hapten (prepared as described in general Scheme 3)proceeds via a Michael addition reaction. Suitable proteins are known tothose skilled in the art and include keyhole limpet hemocyanin, bovinethyroglobulin, and ovalbumin. While Scheme 8 illustrates protein-haptenconjugation where R¹ is

the same chemistry can be used to conjugate any maleimide functionalizedhapten to a protein.

-   -   where x is m or n, as defined in Formula I.

Carboxylic acid functionalized haptens may be conjugated to proteinsaccording to the method shown in Scheme 9. Reaction withN-hydroxysuccinimide and a suitable coupling agent, such asdicyclohexylcarbodiimide (DCC) and a base such as tributylamine, in asolvent such as DMF at a temperature of about 20° C., for about 18hours, activates the carboxylic acid with thehydroxypyrrolidine-2,5-dione leaving group. The activated spacer andhapten may then be conjugated to a protein in a solvent, such as pH 7.5phosphate buffer, at about 20° C. for about 2.5 hours. Suitable proteinsare known to those skilled in the art and include keyhole limpethemocyanin, bovine thyroglobulin, and ovalbumin. While Scheme 9illustrates protein-hapten conjugation where R² is NHC(O)(CH₂)_(n)CO₂H,the same chemistry can be used to conjugate any CO₂H functionalizedhapten to a protein.

Antibody Production

The conjugates above are useful for the production of antibodies whichbind the anti-psychotic drug to which they were generated(aripiprazole). These antibodies can be used in assays to detect thepresence and/or amount of the anti-psychotic drug in patient samples.Such detection permits therapeutic drug monitoring enabling all of thebenefits thereof. Detection of levels of anti-psychotic drugs may beuseful for many purposes, including: detection in combination with thedetection of other anti-psychotic drugs, including those selected fromthe group consisting of risperidone, paliperidone, quetiapine,olanzapine, and metabolites thereof, such detection permitting thesimultaneous measurement of these anti-psychotic drugs; determination ofpatient adherence or compliance with prescribed therapy; use as adecision tool to determine whether a patient should be converted from anoral anti-psychotic regimen to a long-acting injectable anti-psychoticregimen; use as a decision tool to determine if the dose level or dosinginterval of oral or injectable anti-psychotics should be increased ordecreased to ensure attainment or maintenance of efficacious or safedrug levels; use as an aid in the initiation of anti-psychotic drugtherapy by providing evidence of the attainment of minimum pK levels;use to determine bioequivalence of anti-psychotic drug in multipleformulations or from multiple sources; use to assess the impact ofpolypharmacy and potential drug-drug interactions; and use as anindication that a patient should be excluded from or included in aclinical trial and as an aid in the subsequent monitoring of adherenceto clinical trial medication requirements.

Having provided the conjugates of the subject invention, which comprisethe compounds herein and an immunogenic carrier, antibodies can begenerated, e.g., polyclonal, monoclonal, chimeric, and humanizedantibodies, that bind to the anti-psychotic drug. Such antibodies thatare particularly contemplated include monoclonal and polyclonalantibodies as well as fragments thereof, e.g., recombinant proteins,containing the antigen-binding domain and/or one or more complementaritydetermining regions of these antibodies. Preferably, the antibody willbind to the drug and any desired pharmacologically active metabolites.By altering the location of the attachment of the immunogenic carrier tothe compounds of the invention, selectivity and cross-reactivity withmetabolites can be engineered into the antibodies. For aripiprazole,cross-reactivity with dehydroaripiprazole may be desirable. Antibodiesmay be generated that detect both aripiprazole and dehydroaripiprazole,or antibodies may be generated that detect each separately (thusdefining the antibody “specific binding” properties). An antibodyspecifically binds one or more compounds when its binding of the one ormore compounds is equimolar or substantially equimolar.

Methods of producing such antibodies comprise inoculating a host withthe conjugate (the compound and the immunogenic carrier being animmunogen) embodying features of the present invention. Suitable hostsinclude, but are not limited to, mice, rats, hamsters, guinea pigs,rabbits, chickens, donkeys, horses, monkeys, chimpanzees, orangutans,gorillas, humans, and any species capable of mounting a mature immuneresponse. The immunization procedures are well established in the artand are set forth in numerous treatises and publications including “TheImmunoassay Handbook”, 2nd Edition, edited by David Wild (NaturePublishing Group, 2000) and the references cited therein.

Preferably, an immunogen embodying features of the present invention isadministered to a host subject, e.g., an animal or human, in combinationwith an adjuvant. Suitable adjuvants include, but are not limited to,Freund's adjuvant, powdered aluminum hydroxide (alum), aluminumhydroxide together with Bordetella pertussis, and monophosphoryl lipidA-synthetic trehalose dicorynomycolate (MPL-TDM).

Polyclonal antibodies can be raised in a mammalian host by one or moreinjections of an immunogen which can optionally be administered togetherwith an adjuvant. Typically, an immunogen or a combination of animmunogen and an adjuvant is injected into a mammalian host by one ormultiple subcutaneous or intraperitoneal injections. Preferably, theimmunization program is carried out over at least one week, and morepreferably, over two or more weeks. Polyclonal antibodies produced inthis manner can be isolated and purified utilizing methods well know inthe art.

Monoclonal antibodies can be produced by the well-established hybridomamethods of Kohler and Milstein, e.g., Nature 256:495-497 (1975).Hybridoma methods typically involve immunizing a host or lymphocytesfrom a host, harvesting the monoclonal antibody secreting or having thepotential to secrete lymphocytes, fusing the lymphocytes to immortalizedcells, and selecting cells that secrete the desired monoclonal antibody.

A host can be immunized to elicit lymphocytes that produce or arecapable of producing antibodies specific for an immunogen.Alternatively, the lymphocytes can be immunized in vitro. If human cellsare desired, peripheral blood lymphocytes can be used, although spleencells or lymphocytes from other mammalian sources are preferred.

The lymphocytes can be fused with an immortalized cell line to formhybridoma cells, a process which can be facilitated by the use of afusing agent, e.g., polyethylene glycol. By way of illustration, mutantrodent, bovine, or human myeloma cells immortalized by transformationcan be used. Substantially pure populations of hybridoma cells, asopposed to unfused immortalized cells, are preferred. Thus, followingfusion, the cells can be grown in a suitable medium that inhibits thegrowh or survival of unfused, immortalized cells, for example, by usingmutant myeloma cells that lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT). In such an instance, hypoxanthine,aminopterin, and thymidine can be added to the medium (HAT medium) toprevent the growth of HGPRT-deficient cells while permitting hybridomasto grow.

Preferably, immortalized cells fuse efficiently, can be isolated frommixed populations by selection in a medium such as HAT, and supportstable and high-level expression of antibody following fusion. Preferredimmortalized cell lines include myeloma cell lines available from theAmerican Type Culture Collection, Manassas, Va.

Because hybridoma cells typically secrete antibody extracellularly, theculture media can be assayed for the presence of monoclonal antibodiesspecific for the anti-psychotic drug. Immunoprecipitation of in vitrobinding assays, for example, radiioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA), can be used to measure the bindingspecificity of monoclonal antibodies.

Monoclonal antibody-secreting hybridoma cells can be isolated as singleclones by limiting dilution procedures and sub-cultured. Suitableculture media include, but are not limited to, Dulbecco's ModifiedEagle's Medium, RPMI-1640, and polypeptide-free, polypeptide-reduced, orserum-free media, e.g., Ultra DOMA PF or HL-1, available fromBiowhittaker, Walkersville, Md. Alternatively, the hybridoma cells canbe grown in vivo as ascites.

Monoclonal antibodies can be isolated and/or purified from a culturemedium or ascites fluid by conventional immunoglobulin (Ig) purificationprocedures including, but not limited to, polypeptide A-SEPHAROSE,hydroxylapatite chromatography, gel electrophoresis, dialysis, ammoniumsulfate precipitation, and affinity chromatography.

Monoclonal antibodies can also be produced by recombinant methods suchas are described in U.S. Pat. No. 4,166,452. DNA encoding monoclonalantibodies can be isolated and sequenced using conventional procedures,e.g., using oligonucleotide probes that specifically bind to murineheavy and light antibody chain genes, preferably to probe DNA isolatedfrom monoclonal antibody hybridoma cells lines secreting antibodiesspecific for anti-psychotic drugs.

Immunoassays

The antibodies thus produced can be used in immunoassays torecognize/bind to the anti-psychotic drug, thereby detecting thepresence and/or amount of the drug in a patient sample. Preferably, theassay format is a competitive immunoassay format. Such an assay formatand other assays are described, among other places, in Hampton et al.(Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn.1990) and Maddox et al. (J. Exp. Med. 158:12111, 1983).

A reagent kit can also be provided comprising an antibody as describedabove. A representative reagent kit may comprise an antibody that bindsto the anti-psychotic drug, aripiprazole, a complex comprising an analogof an anti-psychotic drug or a derivative thereof coupled to a labelingmoiety, and may optionally also comprise one or more calibratorscomprising a known amount of an anti-psychotic drug or a relatedstandard.

As noted above, reagent kits may comprise calibrators and/or controlmaterials which comprise a known amount of the analyte to be measured.The concentration of the analyte can be calculated by comparing resultsobtained for a sample with resulted obtained for a standard. Acalibration curve can be constructed and used for relating the sets ofresults and for determining the concentration of an analyte in a sample.

Any sample that is suspected of containing an analyte, e.g., ananti-psychotic drug, can be analyzed in accordance with the methods ofthe presently preferred embodiments. The sample can be pretreated ifdesired and can be prepared in any convenient medium that does notinterfere with the assay. Preferably, the sample comprises an aqueousmedium such as a body fluid from a host, most preferably plasma orserum.

Copending applications entitled “Haptens of Aripiprazole” (AttorneyDocket No. PRD3265USPSP/USNP, U.S. Provisional Patent Appl. No.61/691,450, filed Aug. 21, 2012, and US 20140163206, filed Aug. 20,2013), “Haptens of Olanzapine” (Attorney Docket No. PRD3266USPSP/USNP,U.S. Provisional Patent Appl. No. 61/691,454, filed Aug. 21, 2012, andUS 20140213766, filed Aug. 20, 2013), “Haptens of Paliperidone”(Attorney Docket No. PRD3267USPSP/USNP, U.S. Provisional Patent Appl.No. 61/691,459, filed Aug. 21, 2012 and US 2014/0213767, filed Aug. 20,2013), “Haptens of Quetiapine” (Attorney Docket No. PRD3268USPSP/USNP,U.S. Provisional Patent Appl. No. 61/691,462, filed Aug. 21, 2012, andUS 20140221616, filed Aug. 20, 2013), “Haptens of Risperidone andPaliperidone” (Attorney Docket No. PRD3269USPSP/USNP, U.S. ProvisionalPatent Appl. No. 61/691,469, filed Aug. 21, 2012, and US 20140155585,Aug. 20, 2013), “Antibodies to Aripiprazole Haptens and Use Thereof”(Attorney Docket No. CDS5128USPSP/USNP, U.S. Provisional Patent Appl.No. 61/691,544, filed Aug. 21, 2012, and US 20140057299, filed Aug. 20,2013), “Antibodies to Olanzapine Haptens and Use Thereof” (AttorneyDocket No. CDS5132USPSP/USNP, U.S. Provisional Patent Appl. No.61/691,572, filed Aug. 21, 2012, US 20140057303, filed Aug. 20, 2013),“Antibodies to Paliperidone Haptens and Use Thereof” (Attorney DocketNo. CDS5126USPSP/USNP, U.S. Provisional Patent Appl. No. 61/691,634,filed Aug. 21, 2012, and US 20140057297, filed Aug. 20, 2013),“Antibodies to Quetiapine Haptens and Use Thereof” (Attorney Docket No.CDS5134USPSP/USNP, U.S. Provisional Patent Appl. No. 61/691,598, filedAug. 21, 2012, and US 20140057305, filed Aug. 20, 2013), “Antibodies toRisperidone Haptens and Use Thereof” (Attorney Docket No.CDS5130USPSP/USNP, U.S. Provisional Patent Appl. No. 61/691,615, filedAug. 21, 2012, and US 20140057301, filed Aug. 20, 2013), “Antibodies toAripiprazole and Use Thereof” (Attorney Docket No. CDS5129USPSP/USNP,U.S. Provisional Patent Appl. No. 61/691,522, filed Aug. 21, 2012, andUS 20140057300, filed Aug. 20, 2013), “Antibodies to Olanzapine and UseThereof” (Attorney Docket No. CDS5133USPSP/USNP, US Provisional PatentAppl. No. 61/691,645, filed Aug. 21, 2012, and US 20140057304, filedAug. 20, 2013), “Antibodies to Paliperidone and Use Thereof” (AttorneyDocket No. CDS5127USPSP/USNP, U.S. Provisional Patent Appl. No.61/691,692, filed Aug. 21, 2012, and US 20140057298, filed Aug. 20,2013), “Antibodies to Quetiapine and Use Thereof” (Attorney Docket No.CDS5135USPSP/USNP, U.S. Provisional Patent Appl. No. 61/691,659, filedAug. 21, 2012, and US 20140057306, filed Aug. 20, 2013), and “Antibodiesto Risperidone and Use Thereof” (Attorney Docket No. CDS5131USPSP/USNP,U.S. Provisional Patent Appl. No. 61/691,675, filed Aug. 21, 2012, andUS 20140057302, filed Aug. 20, 2013) are all incorporated herein byreference in their entireties.

EXAMPLES

Representative compounds of the present invention can be synthesized inaccordance with the general synthetic methods described below. Compoundsof Formula (I) can be prepared by methods known to those who are skilledin the art. The following examples are only meant to represent examplesof the invention and are in no way meant to be a limit of the invention.

Example 14-(aminomethyl)-7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one

Step A 1-(bromomethyl)-4-methoxy-2-nitrobenzene

To a well-stirred solution of compound 4-methoxy-1-methyl-2-nitrobenzene(218 g, 1.30 mol) in CCl₄ (1500 mL) was added AIBN (21.7 g, 0.13 mol),and NBS (348 g, 1.96 mol). After the reaction mixture was heated atreflux for 16 h under N₂, water was added and the product extracted fromthe aqueous phase with CH₂Cl₂. The resultant organic phase was washedwith brine, dried over Na₂SO₄ and the solvent was evaporated to give asolid which was purified by silica gel chromatography (eluting withpetroleum ether/ethyl acetate, 20:1) to the title compound as a yellowsolid. ESI-MS (M+1) 246. ¹H NMR: (CDCl₃, 400 MHz): δ (ppm) 7.55 (s, 1H),7.46-7.42 (d, 1H), 7.14-7.11 (d, 1H), 4.79 (s, 2H), 3.90 (s, 3H).

Step B 2-(4-methoxy-2-nitrophenyl)acetonitrile

To a stirred solution of 1-(bromomethyl)-4-methoxy-2-nitrobenzene,prepared as described in Step A, (40 g, 0.163 mol) in THF (500 mL) andEtOH (100 mL) was added a solution of KCN (26.6 g, 0.408 mol) in water(100 mL). The reaction mixture was stirred at 0° C. for 1 h and thenfurther for 3 h at room temperature. The reaction mixture was dilutedwith water (500 mL) and aqueous phase was extracted with DCM (500 mL)and then washed with brine, dried over Na₂SO₄ and evaporated in vacuo.The residue was purified by chromatography on a silica gel column togive the title compound. ESI-MS (M+1) 193. ¹H NMR: (CDCl₃, 400 MHz): δ(ppm) 7.72 (s, 1H), 7.63-7.61 (d, 1H), 7.26-7.23 (d, 1H), 4.14 (s, 2H),3.93 (s, 3H).

Step C ethyl 3-cyano-3-(4-methoxy-2-nitrophenyl)propanoate

To a solution of 2-(4-methoxy-2-nitrophenyl)acetonitrile, prepared asdescribed in Step B, (5.5 g, 0.0286 mol) in DMF (100 mL) was addedBrCH₂CO₂Et (5.71 g, 0.034 mol) and K₂CO₃ (11.86 g, 0.086 mol) at 0° C.The reaction mixture was stirred at 0° C. for 1 h and at roomtemperature for another 2 h. After the reaction was completed by TLCmonitoring, water was added. The reaction was extracted with ethylacetate; the organic phase was washed with brine, dried over Na₂SO₄, andconcentrated in vacuo. The crude product was purified by chromatographyon a silica gel column to give the title compound. ESI-MS (M+1) 279. ¹HNMR: (CDCl₃, 400 MHz): δ (ppm) 7.70-7.68 (d, 1H), 7.57-7.56 (s, 1H),7.24-7.21 (d, 1H), 5.13-4.98 (m, 1H), 4.20-4.18 (m, 2H), 3.89 (s, 3H),2.99-2.97 (d, 2H), 1.28-1.24 (t, 3H).

Step D 7-methoxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carbonitrile

To a solution of ethyl 3-cyano-3-(4-methoxy-2-nitrophenyl)propanoate,prepared as described in Step C, (9.0 g, 0.032 mol) in MeOH (100 mL), Sn(19.3 g, 0.162 mol) was added, followed by hydrochloric acid/MeOH (40ml, 1:1) all at once. The reaction was stirred at room temperature for 2h. The solvent was removed in vacuo. Then ethyl acetate was added, andaqueous NaHCO₃ solution was added to neutralize the solution. Theorganic phase was concentrated to get crude product which was used fornext step without further purification.

Step E 4-(aminomethyl)-7-methoxy-3,4-dihydroquinolin-2(1H)-one

Crude 7-methoxy-2-oxo-1,2,3,4-tetrahydroquinoline-4-carbonitrile,prepared as described in Step D, (6 g, 0.03 mol) and Raney Ni (10 g) wassuspended in a mixture of MeOH (100 mL) and 3 mL of triethylamine. Thereaction mixture was stirred under H₂ (50 Psi) atmosphere at roomtemperature for 4 h. After the reaction was completed by monitoring byTLC, the catalyst was filtered off, and then the solvent was removed invacuo to afford the crude product which was used for next step withoutfurther purification.

Step F 4-(aminomethyl)-7-hydroxy-3,4-dihydroquinolin-2(1H)-one

To a solution of crude4-(aminomethyl)-7-methoxy-3,4-dihydroquinolin-2(1H)-one, prepared asdescribed in Step E, (8.8 g, 0.0427 mol) in dichloromethane (100 mL),BBr₃ (85 g, 0.342 mol) in dichloromethane (IM) was added dropwise at−14° C., and the reaction was stirred at room temperature overnight.After the reaction was completed by monitoring through TLC, methanol wasadded slowly at 0° C. to quench the reaction, and the solvent wasevaporated in vacuo to get crude product which was used directly in thenext step.

Step G tert-butyl((7-hydroxy-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)carbamate

Crude 4-(aminomethyl)-7-hydroxy-3,4-dihydroquinolin-2(1H)-one, preparedas described in Step F, (8.2 g, 0.0427 mol) and (Boc)₂O (4.65 g, 0.021mol), triethylamine (10 mL) were added to 100 mL of methanol. Thereaction was stirred at room temperature for 2 h. After the reaction wasstopped, the solvent was removed in vacuo, and ethyl acetate was added.The organic phase was washed with water, aqueous NaHCO₃ solution, driedover Na₂SO₄ and concentrated in vacuo. The crude product was purified bychromatography to give the title compound. ESI-MS (M+1) 292. ¹H NMR:(DMSO-d₆, 400 MHz): δ (ppm) 9.96 (s, 1H), 9.31 (s, 1H), 6.95-6.89 (m,2H), 6.33 (d, 2H), 3.00-2.97 (m, 2H), 2.90-2.96 (m, 1H), 2.56 (m, 1H),2.30-2.34 (m, 1H), 1.37 (s, 9H).

Step H tert-butyl((7-(4-bromobutoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)carbamate

To a solution of tert-butyl((7-hydroxy-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)carbamate,prepared as described in Step H, (1.0 mmol, 292 mg) and1,4-dibromobutane (1.1 mmol, 237.5 mg) in DMF (1.5 mL) was addedanhydrous K₂CO₃ (1.2 mmol, 166 mg). The mixture was stirred at roomtemperature overnight until HPLC and LC/MS indicated that the reactionwas complete to give the title compound, which was subjected to nextreaction without purification. MS m/z 428 (MH⁺).

Step I tert-butyl((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)carbamate

To a solution of tert-butyl((7-(4-bromobutoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)carbamate,prepared as described in Step H, in DMF was added1-(2,3-dichloro-phenyl)-piperazine hydrochloride (1.0 mmol, 268 mg) andK₂CO₃ (1.23 mmol, 170 mg). The mixture was stirred at room temperatureovernight. The solvent was evaporated in vacuo and the residue waspartitioned between dichloromethane and saturated aqueous NaHCO₃solution. The organic layer was separated and aqueous layer wasextracted with additional dichloromethane. Organic layers were combined,concentrated. The residue was then subjected to column chromatography onsilica gel with gradient 0-10% methanol in dichloromethane to give thetitle compound as a solid. MS m/z 578 (MH⁺).

Step J4-(aminomethyl)-7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one

To a solution of tert-butyl((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)carbamate,prepared as described in Step I, (200 mg, 0.35 mmol) in dichloromethane(5 mL) was added 1 mL of TFA. The mixture was stirred at roomtemperature for 2.5 hr. The solvent was evaporated in vacuo and theresidue was partitioned between dichloromethane and saturated NaHCO₃solution. The organic layer was separated and aqueous layer wasextracted with additional dichloromethane. The organic layers werecombined, dried over Na₂SO₄, filtered and concentrated. The residue wasthen subjected to column chromatography on silica gel with 10% methanolin dichloromethane, followed by 10% 7N ammonia methanol indichloromethane, to give the title compound as a solid. This product wasfurther purified by recrystalization from dichloromethane and heptanesto give final product as a white solid. MS m/z 477 (MH⁺). ¹H NMR:(CDCl₃, 400 MHz): δ (ppm) 7.40 (s, 1H), 7.25-7.05 (m, 3H), 7.00 (d, 1H),6.60 (d, 1H), 6.30 (s, 1H), 4.00 (m, 2H), 3.10 (m, 4H), 3.00-2.60 (m,9H), 2.50 (m, 2H), 1.90-1.40 (m, 6H). Calculated for C₂₄H₃₀Cl₂N₄O₂ C,60.38; H, 6.33; N, 11.74. Found C, 60.32; H, 5.89; N, 11.26.

Example 27-(4-(4-(4-amino-2,3-dichlorophenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one

Step A 4-bromo-2,3-dichloro-N-(4-methoxybenzyl)aniline

To a solution of 4-bromo-2,3-dichloro-phenylamine (3.215 g, 13.3 mmol)and 1-chloromethyl-4-methoxy-benzene (2.297 g, 14.7 mmol) in 23 mL ofDMF was added potassium iodide (2.214 g, 13.3 mmol) and 647 mg of sodiumhydride (60% oil dispersion). After stirring at room temperatureovernight, the reaction mixture was evaporated in vacuo and the residuewas partitioned between dichloromethane and saturated NaHCO₃ aqueoussolution. The organic layer was separated and aqueous layer wasextracted with additional dichloromethane. The organic layers werecombined, dried over Na₂SO₄, filtrated, and concentrated. The residuewas then subjected to column chromatography on silica gel with 30% ethylacetate in heptanes to give the title compound as a yellow solid; MS m/z362 (MH⁺).

Step B 2,3-dichloro-N-(4-methoxybenzyl)-4-(piperazin-1-yl)aniline

A mixture of 4-bromo-2,3-dichloro-N-(4-methoxybenzyl)aniline, preparedas described in the previous step, (3.61 g, 10 mmol), piperizine (1.034g, 12 mmol), sodium t-butoxide (1.16 g, 12 mmol), andtris(dibenzylideneacetone) dipalladium(0) (180 mg, 2 mol %) in 16 mL oftoluene in a sealed thick-wall flask was stirred and heated in an oilbath at 100° C. for 2.5 days. After cooling to room temperature, thereaction mixture was partitioned between dichloromethane and water. Theorganic layer was separated and aqueous layer was extracted withadditional dichloromethane. The organic layers were combined, dried overNa₂SO₄, filtered, and concentrated. The residue was purified by columnchromatography on silica gel column with 10% methanol indichloromethane, followed by 10% 7N ammonia methanol in dichloromethane,to give the title compound as a light brown solid. MS m/z 367 (MH⁺). ¹HNMR: (CDCl₃, 400 MHz): δ (ppm) 7.28 (d, 2H), 6.98 (d, 2H), 6.50 (d, 1H),4.60 (s, 1H), 4.30 (m, 2H), 3.80 (m, 3H), 3.10-2.85 (m, 8H), 2.30 (s,1H).

Step C 2,3-dichloro-4-(piperazin-1-yl)aniline

To a solution of2,3-dichloro-N-(4-methoxybenzyl)-4-(piperazin-1-yl)aniline, prepared asdescribed in the previous step, (562 mg, 1.54 mmol) in dichloromethane(5 mL) was added 5 mL of TFA. The reaction mixture was stirred at roomtemperature for 5 hr, and then was evaporated in vacuo to dryness. Theresidue was re-dissolved in dichloromethane and evaporated to dryness.The title compound was used in the next reaction without purification.MS m/z 245 (MH⁺).

Step D7-(4-(4-(4-amino-2,3-dichlorophenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one

To a solution of 2,3-dichloro-4-(piperazin-1-yl)aniline, prepared asdescribed in the previous step, (1.535 mmol) as TFA salt in DMF (6 mL)was added a solution of commercially available7-(4-bromo-butoxy)-3,4-dihydro-1H-quinolin-2-one (1.535 mmol) in DMF (1mL), K₂CO₃ (2.121 g), and 1 mL of DMF. The resultant mixture was stirredat room temperature overnight. The solid was filtered and rinsed withdichloromethane. The solution was evaporated in vacuo and the residuewas then subjected to column chromatography on silica gel with gradient0-10% methanol in dichloromethane, followed by 10% 7N ammonia methanolin dichloromethane, to give the title compound as a solid. MS m/z 463(MH⁺). ¹H NMR: (DMSO-d₆, 400 MHz): δ (ppm) 10.0 (s, 1H), 7.05 (d, 1H),6.95 (d, 1H), 6.75 (d, 1H), 6.50 (d, 1H), 6.45 (s, 1H), 5.3 (s, 2H),3.90 (m, 2H), 2.90-2.70 (m, 6H), 2.50-2.30 (m, 8H), 1.80-1.50 (m, 4H).Calculated for C₂₃H₂₈Cl₂N₄O₂ is C, 59.61; H, 6.09; N, 12.09. Found C,59.44; H, 5.87; N, 11.77.

Example 34-((2,3-dichloro-4-(4-(4-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy)butyl)piperazin-1-yl)phenyl)amino)-4-oxobutanoicacid

A solution of Example 2 (115.5 mg, 0.25 mmol) and succinic anhydride (50mg, 0.5 mmol) in pyridine (1.5 mL) was stirred and heated at 110° C. ina microwave oven for 5.5 hr. The solution was evaporated in vacuo todryness. The residue was re-dissolved in dichloromethane and evaporatedto dryness; and then re-dissolved in methanol and evaporated to dryness.The crude product was purified on a Agela hilic column with gradient0-20% methanol in dichloromethane to give a solid, which was furtherpurified by recrystalization from methanol and dried at 40-50° C. in avacuum oven to give the title compound. MS m/z 563 (MH⁺). ¹H NMR:(DMSO-d₆, 400 MHz): δ (ppm) 12.1 (s, 1H), 10.0 (s, 1H), 9.60 (s, 1H),7.5 (d, 1H), 7.15 (d, 1H), 7.05 (d, 1H), 6.50 (d, 1H), 6.45 (s, 1H),3.90 (m, 2H), 3.00 (m, 4H), 2.30 (m, 2H), 2.20-2.30 (m, 12H), 1.80-1.55(m, 4H). Calculated for C₂₇H₃₃Cl₂N₄O₅ is C, 57.55; H, 5.72; N, 9.94.Found C, 55.92; H, 5.85; N, 9.58.

Example 45-((2,3-dichloro-4-(4-(4-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy)butyl)piperazin-1-yl)phenyl)amino)-5-oxopentanoicacid

A solution of Example 2 (83 mg, 0.18 mmol) and glutaric anhydride (41mg, 0.36 mmol) in pyridine (1.0 mL) was stirred and heated at 110° C. ina microwave oven for 4.5 hr. The solution was evaporated in vacuo todryness. The residue was purified on an Agela hilic column (12 g) withgradient 0-30% methanol and dried at 40-50° C. in a vacuum oven to givethe title compound. MS m/z 578 (MH⁺).

Example 54-(((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)amino)-4-oxobutanoicacid

A solution of Example 1 (24.1 mg, 0.05 mmol) and succinic anhydride (10mg, 0.10 mmol) in THF (1.0 mL) was stirred at room temperatureovernight. The solution was evaporated in vacuo to dryness. The residuewas purified on a silica gel column (12 g) with gradient 0-30% methanolin dichloromethane and dried at 40-50° C. in a vacuum oven to give thetitle compound. MS m/z 578 (MH⁺).

Example 6N-((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide

To a solution of Example 1 (MW 477.4 2.2 mg, 4.61 moles) in 110 μL ofDMF and 2.3 μL of tributylamine was added 116 μL of a DMF solution ofN-(α-maleimidoacetoxy) succinimide ester (AMAS, MW 252.2, 10 mg/mL, 1.16mg, 4.61 moles). The resulting solution was allowed to stir for 90minutes at 20° C., and then used as such in conjugation reaction withthiol-activated protein.

Example 7N-((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide-keyholelimpet hemocyanin conjugate

To 3.23 mL of a solution of keyhole limpet hemocyanin (KLH MW 100,00014.6 mg, 0.146 μmoles) in 100 mM phosphate buffer, 0.46M sodiumchloride, pH 7.4 was added 33.7 μL of a DMF solution ofN-Succinimidyl-S-acetylthioacetate (SATA MW 231.2, 25 mg/mL, 0.84 mg,3.65 μmoles). The resulting solution was incubated at 20° C. for 1 houron a roller mixer. The reaction was purified on a Sephadex G-25 columnusing 100 mM phosphate buffer, 0.46M sodium chloride, and 5 mM EDTA, atpH 6.0. To 6.46 mL of the KLH-SATA solution (13.7 mg, 0.137 μmoles) wasadded 646 μL of 2.5M hydroxylamine, and 50 mM EDTA, at pH 7.0. Theresulting solution was incubated at 20° C. for 1 hour on a roller mixer.The reaction was treated with 169.6 μL ofN-((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamidesolution (prepared as described in example 6) (3.43 μmoles). Theresulting cloudy mixture was incubated for 2 hours at 20° C. on a rollermixer. The reaction was filtered through a 0.2 am syringe filter thenpurified on a Sephadex G-25 column using 100 mM phosphate buffer and0.46M sodium chloride at pH 7.4, to give the KLH conjugate of Example 6.

Example 8N-((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)acetamide-bovinethyroglobulin conjugate

To 2.14 mL of a solution of bovine thyroglobulin (BTG, MW 660,000, 21.8mg, 0.033 μmoles) in a 100 mM phosphate buffer at pH 7.5 was added 61.1μL of a DMF solution of N-succinimidyl-S-acetylthioacetate (SATA, MW231.2, 25 mg/mL, 1.53 mg, 6.6 μmoles). The resulting solution wasincubated at 20° C. for 1 hour on a roller mixer. The reaction waspurified on a Sephadex G-25 column using 100 mM phosphate buffer, 5 mMEDTA, at pH 6.0. To 5.79 mL of BTG-SATA (20.5 mg, 0.031 μmoles) wasadded 579 μL of 2.5M hydroxylamine, and 50 mM EDTA, at pH 7.0. Theresulting solution was incubated at 20° C. for 1 hour on a roller mixer.The reaction was treated with 304.0 μL ofN-((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamidesolution (prepared as described in example 6) (6.2 μmoles). Theresulting cloudy mixture was incubated for 2 hours at 20° C. on a rollermixer. The reaction was filtered through a 0.45 μm syringe filter thenpurified on a Sephadex G-25 column using 100 mM phosphate buffer and0.14M sodium chloride at pH 7.4, to give the bovine thyroglobulinconjugate of Example 6.

Example 9N-((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide-ovalbuminconjugate Step A

To 1.0 mL of a solution of ovalbumin (MW 44,300, 10.0 mg, 0.23 μmoles)in 100 mM phosphate buffer at pH 7.5 was added 31.3 μL of a DMF solutionof N-Succinimidyl-S-acetylthioacetate (SATA, MW 231.2, 25 mg/mL, 0.78mg, 3.4 μmoles). The resulting solution was incubated at 20° C. for 1hour on a roller mixer. The reaction was treated with 100 μL of 2.5Mhydroxylamine and 50 mM EDTA at pH 7.0. The resulting solution wasincubated at 20° C. for 15 minutes on a roller mixer. The reaction waspurified on a Sephadex G-25 column using 100 mM phosphate buffer and 5mM EDTA at pH 6.0.

Step B

To the ovalbumin-SH, (3.1 mL, 8.3 mg, 0.187 μmol), prepared as describedin Step A, was addedN-((7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2-oxo-1,2,3,4-tetrahydroquinolin-4-yl)methyl)-2-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)acetamidesolution (prepared as described in example 6) ((185.7 μL, 3.75 μmoles).The resulting cloudy mixture was incubated for 2.5 hours at 20° C. on aroller mixer. The reaction was filtered through a 0.45 am syringefilter, then purified on a Sephadex G-25 column using 100 mM phosphatebuffer, 0.14M sodium chloride at pH 7.4, to give the ovalbumin conjugateof Example 6.

Example 10 Competitive Immunoassay for Aripiprazole

Following a series of immunizations with the immunogens described abovein Examples 7-9, mouse tail bleeds were tested for reactivity using anELISA. Hybridoma supernatants were also tested, and the ELISA data shownin Table 8 below shows reactivity of several hybridomas (fusion partnerwas NSO cells).

TABLE 8 Plate 1 Dilution 1 2 3 4 5 400 3C1 3D7 5C6 5C7 5H11 400 12001200 3600 3600 10800 10800 400 0.8165 0.7299 0.196 3.2953 0.0373 4000.7057 0.5671 0.1525 2.9591 0.0371 1200 0.2413 0.2186 0.0701 1.92420.0348 1200 0.2474 0.2278 0.0653 1.7829 0.0336 3600 0.102 0.0963 0.04720.739 0.0288 3600 0.099 0.0954 0.051 0.7225 0.0281 10800 0.0534 0.05260.0381 0.2878 0.0215 10800 0.0644 0.0588 0.0411 0.2799 0.0326

Supernatant was then tested by competition ELISA to determine if thesignal was specific to either aripiprazole or dehydroaripiprazole. FIGS.1 and 2 show the results from two representative hybridomas, 3C1 and3D7. Data shows reactivity to both aripiprazole and dehydroaripiprazole.

FIG. 3 shows the competitive immunoassay format used on a lateral flowassay device in which the capture antibody, aripiprazole clone 5C7, wasdeposited on a chip along with a detection conjugate consisting ofaripiprazole conjugated to a fluorophore. In this competitive format asshow in FIG. 3, a low level of analyte (aripiprazole) results in highsignal, whereas a high level of analyte (aripiprazole) results in lowsignal. Referring to FIGS. 4 and 5 which show the results from the assayas run on a lateral flow assay device, as the dose of aripiprazole inthe sample increased, it competed for binding sites on the antibodies.The amount of aripiprazole in the sample can thus be calculated from theloss in fluorescence compared to a sample with no drug present.

All documents cited herein are incorporated by reference. While theforegoing specification teaches the principles of the present invention,with examples provided for the purpose of illustration, it will beunderstood that the practice of the invention encompasses all of theusual variations, adaptations and/or modifications as come within thescope of the following Claims and their equivalents.

We claim:
 1. A conjugate of the compound of Formula I and an immunogeniccarrier:

wherein: R¹ is H

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

NH₂, or NHC(O)(CH₂)_(m)CO₂H; R³ is H; provided that either R¹ or R² mustbe H, and further provided that R¹ and R² may not be H simultaneously; mis 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or
 5. 2. The conjugate ofclaim 1 wherein: R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

NH₂, or NHC(O)(CH₂)_(m)CO₂H; provided that either R¹ or R² must be H,and further provided that both R¹ and R² may not be H simultaneously; R³is H; m is 1, 2, 3 or 4; and n is 1, 2, 3, 4, or
 5. 3. The conjugate ofclaim 1 wherein: R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

NH₂, or NHC(O)(CH₂)_(m)CO₂H; R³ is H, provided that either R¹ or R² mustbe H, and further provided that both R¹ and R² may not be Hsimultaneously; m is 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or
 5. 4. Theconjugate of claim 1 wherein: R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H; provided that either R¹ or R² must be H, andfurther provided that both R¹ and R² may not be H simultaneously; R³ isH; m is 1, 2, or 3; and n is 1, 2, or
 3. 5. The conjugate of claim 1,wherein the compound is


6. The conjugate of claim 5, wherein the immunogenic carrier is aprotein.
 7. The conjugate of claim 6, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.
 8. The conjugateof claim 1, wherein the compound is


9. The conjugate of claim 8, wherein the immunogenic carrier is aprotein.
 10. The conjugate of claim 9, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.
 11. The conjugateof claim 1, wherein the compound is


12. The conjugate of claim 11, wherein the immunogenic carrier is aprotein.
 13. The conjugate of claim 12, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.
 14. The conjugateof claim 1, wherein the compound is

wherein m is 2 or
 3. 15. The conjugate of claim 14, wherein theimmunogenic carrier is a protein.
 16. The conjugate of claim 15, whereinthe protein is keyhole limpet hemocyanin, bovine thyroglobulin, orovalbumin.
 17. The conjugate of claim 1, wherein the compound is


18. The conjugate of claim 17, wherein the immunogenic carrier is aprotein.
 19. The conjugate of claim 18, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.
 20. The conjugateof claim 1, wherein the compound is


21. The conjugate of claim 20, wherein the immunogenic carrier is aprotein.
 22. The conjugate of claim 21, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.
 23. A productmade by the process of contacting a compound of Formula I with animmunogenic carrier:

wherein: R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

NH₂, or NHC(O)(CH₂)_(m)CO₂H; R³ is H; provided that either R¹ or R² mustbe H, and further provided that R¹ and R² may not be H simultaneously; mis 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or
 5. 24. The product of claim23, wherein: R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

NH₂, or NHC(O)(CH₂)_(m)CO₂H; provided that either R¹ or R² must be H,and further provided that both R¹ and R² may not be H simultaneously; R³is H; m is 1, 2, 3 or 4; and n is 1, 2, 3, 4, or
 5. 25. The product ofclaim 23, wherein: R¹ is H,

CH₂NH₂, or CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

NH₂, or NHC(O)(CH₂)_(m)CO₂H; R³ is H, provided that either R¹ or R² mustbe H, and further provided that both R¹ and R² may not be Hsimultaneously; m is 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or
 5. 26.The product of claim 23, wherein: R¹ is H,

CH₂NH₂, CH₂NHC(O)(CH₂)_(m)CO₂H; R² is H,

NH₂, NHC(O)(CH₂)_(m)CO₂H; provided that either R¹ or R² must be H, andfurther provided that both R¹ and R² may not be H simultaneously; R³ isH; m is 1, 2, or 3; and n is 1, 2, or
 3. 27. The product of claim 23,wherein the compound is


28. The product of claim 27, wherein the immunogenic carrier is aprotein.
 29. The product of claim 28, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.
 30. The productof claim 23, wherein the compound is


31. The product of claim 30, wherein the immunogenic carrier is aprotein.
 32. The product of claim 31, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.
 33. The productof claim 23, wherein the compound is


34. The product of claim 33, wherein the immunogenic carrier is aprotein.
 35. The product of claim 34, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.
 36. The productof claim 23, wherein the compound is

wherein m is 2 or
 3. 37. The product of claim 36, wherein theimmunogenic carrier is a protein.
 38. The product of claim 37, whereinthe protein is keyhole limpet hemocyanin, bovine thyroglobulin, orovalbumin.
 39. The product of claim 23, wherein the compound is


40. The product of claim 39, wherein the immunogenic carrier is aprotein.
 41. The product of claim 40, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.
 42. The productof claim 23, wherein the compound is


43. The product of claim 42, wherein the immunogenic carrier is aprotein.
 44. The product of claim 43, wherein the protein is keyholelimpet hemocyanin, bovine thyroglobulin, or ovalbumin.